Recombinant vectors for expression of animal free milk protein and process for preparing the animal free milk protein

A method for the production of animal-free milk proteins in recombinant host cells, the method comprising the steps of:

• i. designing plasmid expression vectors comprising a polynucleotide sequence encoding a polypeptide sequence encoding the animal free milk protein having 90% similarity to SEQ ID No. 1 operably linked to promoter by synthesis of gene segments and multiplication of gene segments using PCR techniques;
• ii. carrying spore isolation and protoplastation of fungal host cell to obtain the competent protoplast;
• iii. combining the recombinant vector with the competent protoplast obtained in step (ii) on ice followed by incubating the nutrient media plate for 2-4 weeks;
• and
• iv. isolating the sporulating clones obtained in step (iii) by analysing the genomic DNA to identify the overexpressing milk protein gene;
• v. inoculating the positive transformed clones identified in step (iv) in PDB media in baffled conical flask and growing them in a shaker at 150-180 rpm at 28-30° C. for 5-7 days;
• vi. collecting the supernatant from the samples cultured in step (v) every 24 hr time interval centrifugating at 5000-8000 rpm for 5 min at 2-4° C. to harvest the animal free milk proteins;
• vii. analysing the supernatant using SDS-PAGE and western blot to confirm the overexpression of beta-casein, followed by isolation and purification of the animal free milk protein.